Hexameric storage proteins during metamorphosis and egg production in the diamondback moth, Plutella xylostella (Lepidoptera)

Journal of Insect Physiology 46 (2000) 951–958 www.elsevier.com/locate/jinsphys

Hexameric storage proteins during metamorphosis and egg production in the diamondback moth, Plutella xylostella (Lepidoptera) Diana E. Wheeler *, Irina Tuchinskaya, Norman A. Buck, Bruce E. Tabashnik Department of Entomology, University of Arizona, Tucson, AZ 85721, USA Received 9 August 1999; accepted 4 October 1999

Abstract As in many Lepidoptera, Plutella xylostella adults do not feed on protein and females must use accumulated reserves to supply vitellogenin synthesis. Storage proteins were quantified in females and males from the late larval stage through day 4 of adult life. The level of storage protein peaked in the early pupal stage, with females having about twice as much as males. In males, the level fell through pupal development and dropped to a trace by one day after eclosion. In females, level of storage proteins fell until eclosion, and then rose dramatically within four hours after the molt to about 2/3 of the original peak level. This post-eclosion increase, which has not been reported previously in insects, suggests that adult females synthesize hexamerins to resequester amino acids. Subsequently, the level of storage proteins fell as vitellogenin appeared and eggs were laid. The ability to synthesize and sequester amino acids as storage proteins during the adult stage has wide-ranging implication for protein management in insects, particularly those that are long-lived and have flexible schedules of reproduction.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Hexamerin; Oogenesis-flight syndrome; Vitellogenin

1. Introduction The lives of insects are divided into distinct stages that are variously specialized for feeding, dispersal and reproduction. In addition, distinctly different larval and adult diets are common in holometabolous insects, and Lepidoptera serve as an excellent example of such a strategy. Essential nutrients obtained in one stage but needed in another must be sequestered and carried across stages until they are mobilized. Similarly, if one specialized stage does not feed or restricts its diet, its activities must be supported by nutrient intake during the stage that does feed. A common means of reserving amino acids in insects is storage proteins. These typically hexameric proteins have been best characterized with respect to their role in metamorphosis (Telfer and Kun-

* Corresponding author. Tel.: +1-520-621-3273; fax: +1-520-6211150. E-mail address: [email protected] (D.E. Wheeler).

kel, 1991). The role of amino acid storage in adults has been largely overlooked. In Lepidoptera, the larval form is often characterized as an herbivorous eating machine. The ability of adults to feed varies, from those species that lack functional mouthparts and consume nothing, to a few that obtain virtually all materials used in egg production as adults. The majority of Lepidoptera fall between the extremes, sequestering a large proportion of the raw materials used in eggs as larva, but supplementing that with the adult diet, which typically consists of nectar. The lack of abundant amino acids in the diet of most adults is striking (Norris, 1934; Labine, 1968; Slansky and Scriber, 1985). Also striking is the requirement for egg production by females, which requires substantial amounts of protein. Clearly, most protein destined for eggs must be obtained during the larval stage and stored until synthesis of yolk proteins begins. In some species, eggs are matured during the pupal stage and egg development is complete by the time of eclosion. However, in many species, synthesis of egg proteins does not begin until after eclosion. We predicted that if adults do not acquire

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protein via feeding and synthesis of yolk proteins begins in the adult stage, that storage proteins, containing amino acids reserved for egg development, will persist in adults until such synthesis begins. To test this hypothesis, we selected the diamondback moth, Plutella xylostella (Plutellidae), a major pest of cruciferous crops worldwide (Talekar and Shelton, 1993). Under favorable laboratory conditions, few females have mature eggs at emergence and most contain them four days later (Hillyer and Thorsteinson, 1969; Pivnick et al., 1990).

We used laboratory strains of diamondback moth (LAB, NO-QA) derived from field populations in Hawaii, USA (Tabashnik et al., 1994). They were reared in incubators at 27°C, 40% humidity, and a 14:10 h light:dark cycle. Mated females laid eggs on tissue covered by a cabbage leaf. To initiate a hatch, wipes bearing eggs were placed on cabbage seedlings. After hatching, feeding larvae were given new plants as needed. Late larvae were collected, sexed (Liu and Tabashnik, 1997), and placed in Petri dishes with cabbage leaves and allowed to develop further. After eclosion, adults were transferred to single sex cages and given free access to water and a 10% solution of honey. Individuals were sampled throughout development and stored at ⫺70°C until analysis. We assigned developmental stages based on behavior, external appearance, and age. Larvae were considered late (LL) if they appeared to be wandering or had just begun to spin. Prepupae (PP) had spun cocoons but had not shed the cuticle of the last larval stage. Early pupae (EP), early to mid pupae (EMP), midpupae (MP) and late pupae (LP) were approximately one, two, three and five days older than prepupae, respectively. Late late pupae (LLP) were within a few hours of molting. Late pupae were, physiologically, pharate adults. Very newly emerged adults (VNEA) were collected immediately after adult eclosion. Newly emerged adults (NEA) were less than 4 h post-eclosion. Day 1, 2, 3, and 4 (D1, D2, D3, D4) adults were ⬍24, 24–48, 48–72, and 72–96 h post-eclosion, respectively.

250 rpm. Samples were then centrifuged at 12,000 g for 20 min at 4°C. Aliquots of the supernatant were mixed with sample loading buffer and applied to gels. To determine if this procedure effectively extracted storage proteins, pupae and newly emerged insects of both sexes were processed. Then the pellets were resuspended in sodium dodecyl sulfate (SDS) sample loading buffer, ground and centrifuged again. The second extraction yielded only small amounts of storage proteins. Both denaturing and non-denaturing conditions were used in polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970). SDS was used as a denaturing agent. Bis-acrylamide, often used in PAGE, was replaced by DATD (N,N⬘-diallyltartardiamide) (1:39) in the stock acrylamide mixture. Pore-limited native gels were run for 2,500 vh at 4°C. Molecular weights of proteins were estimated with reference to standards in the high molecular weight calibration kit (Biorad) for SDS gels and electrophoresis molecular weight standards kit (Pharmacia) for native gels. Two-dimensional gel electrophoresis, with non-denaturing conditions in the first dimension and SDS-denaturing conditions in the second, was carried out as described in Martinez and Wheeler (1991) and Wheeler and Buck (1995). Twodimensional gel electrophoresis aids in determining the relationship between a native protein and its subunits and in better separation of subunits of similar molecular size. For quantitative determination of storage proteins, samples were prepared as described above. The gels were scanned at 633 nm using a laser densitometer (LKB Ultrascan XL). Standard curves were generated for each experiment using bovine serum album (BSA) of known quantities from 0.2–3.5 µg. These quantities produced a linear standard curve. The loading of samples was adjusted so that the relevant protein bands were at concentrations that fell on the linear standard curve. Internal standards of 1.0 and 3.0 µg BSA were included on each gel run for quantitative analysis to correct for gel to gel variation. Since the storage proteins do not separate well on SDS gels, the densities of all bands in this region were totaled to give an estimate of the amount of storage protein present at different stages in development and in the two sexes. The small subunit of lipophorin also migrates in this region, but the contribution to the total is quite small. We tested six to 25 insects from each stage.

2.2. Sample preparation and electrophoresis

2.3. Western blots

Samples were disrupted in Tris-buffered saline (20 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) containing the following protease inhibitors: 0.7 µM pepstatin A, 8 µl chymostatin, 10 µM aprotinin and 1 mM AEBSF. Insects were ground in 1.5 ml microfuge tubes with a pestle, first by hand, and then using a pestle attached to a rotating shaft attached to a variable speed motor. Each specimen was ground for 15–20 s at about

Antibodies to several Lepidopteran hexamerins were tested for cross-reactivity. A polyclonal antibody against arylphorin from Manduca sexta was a gift from J.H. Law (see Ryan et al., 1984). M.L. Pan and W.H. Telfer (1996) provided antibodies against two high-methionine (HM) hexamerins isolated from Hyalophora cecropia. P. xylostella hexamerins separated by PAGE were blotted onto nitrocellulose paper. After blotting, the paper was rinsed

2. Materials and methods 2.1. Insects

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thoroughly, air dried, and stored at ⫺20°C. The blots were probed with the primary antibodies (1:1,000). Alkaline phosphatase conjugate served as the detection agent. 2.4. Amino acid analysis Proteins separated by 2-dimensional electrophoresis, as shown in Fig. 3, were transferred to PVDF (polyvinylidene difluoride) membrane (Problott). The core of protein blots were excised and analyzed by the University of Arizona Biotechnology Core Facility, which used an Applied Biosystems Model 420A Amino Acid Analyzer.

3. Results 3.1. Characterization of the storage proteins Three proteins were identified as hexamerins, based on their relative mobilities in native and SDS–PAGE and their affinity for antibodies against known hexamerins in other lepidopteran species. In native PAGE (Fig. 3A), extracts of early female pupae resolved as four bands of protein with weights of 592, 530, 496, and 444 kDa. The 592 kDa protein was identified as lipophorin, based on its relative mobility and its production of a large and small subunit in the second dimension (not shown). The two intermediate-sized proteins were recognized by the two Cecropia antibodies to HM hexamerins. The 444 kDa protein was recognized by antibody to Manduca sexta arylphorin. Amino acid analysis of this protein confirmed it was an arylphorin, with 15.1% of total amino acids made up of phenylalanine and tyrosine. Hexamerins resolved in the first dimension on native PAGE (Fig. 3B) and then subjected to an SDS–PAGE second dimension (Fig. 3C) were also probed with antibodies. The three proteins showed the same affinities for antibodies as they did in native PAGE. In simple SDS–PAGE, hexamerin subunits were similar in size and therefore migrated to similar positions. Different SDS–PAGE protocols yielded slightly differ-

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ent patterns of banding. In large format gels (6×8 in) (Fig. 1), three bands were well resolved and show a relative mobility of about 80 kDa. The wide middle band contained two co-migrating subunits, cross-reacting with antibodies to both arylphorin and HMs. The band with the smallest kDa also cross-reacted with antibody to the HMs of H. cecropia, especially M-Mth (Pan and Telfer, 1996). The identity of the top band was not determined. The molecular weights estimated by native and SDS– PAGE for the holoprotein and subunits, respectively, are consistent with a hexameric structure. 3.2. Pupal development in males and females Storage protein pattern varied with sex (Fig. 1). The hexamerin in lowermost band, an HM hexamerin, was less abundant in males than in females. In addition, the proportion of HM hexamerin was greater in young adult females than early pupae. This indicated that females used arylphorin preferentially during metamorphosis, in comparison to the HM hexamerins. The differences by sex and stage are also shown in the small format gels (3×4 in) in Fig. 2. Size of adults varies among both males and females, and adult females tend to be larger than adult males. For example, in a sample of 56 males and females, newly emerged females were significantly heavier (412±15 µg) than males (297±14 µg) (P⬍0.001, t-test). As size of newly emerged adults increased, the percentage of their prepupal weight retained also increased (Fig. 4), from about 45% up to 70%. In other words, larger individuals lost proportionally less weight during metamorphosis than smaller individuals. The same linear relationship held for both males and females, indicating that males and females follow the same general rule for storage. The fact that females were typically larger than males means that females lose proportionally less weight during metamorphosis than males. Females on average retained 61.0(+1.3)% and males 52.7(+1.3)% (t-test, P⬍0.001). This allometric pattern indicates that larger individuals allocate more to reserves than is required for metamorphosis. 3.3. Patterns of hexamerin accumulation and depletion

Fig. 1. SDS–PAGE showing bands of hexamerin subunits. Relative quantities vary with sex and stage. The loadings for male early pupae, female early pupae, and day 1 adults were different (5, 2.5, and 10% of an individual moth, respectively), so only proportional differences within individuals can be assessed directly from these gels. The two dots on the right indicate molecular weight standards of 66 and 97 kDa.

PAGE indicated that storage protein levels in both females and males peaked in the early pupal stage (Fig. 2). A significant amount of storage protein remained in females after eclosion. In contrast, males were virtually depleted of these proteins. Quantitative gel electrophoresis showed that both weight-specific level of the storage protein, as well as total amount per individual, differed markedly between females and males (Fig. 5). The weight-specific concentration of storage proteins during the early pupal stage peaked at about 28 µg/mg

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Fig. 2. Developmental changes in males and females, from the late larval stage to nearly emerged adults, in the level of storage protein (SDS– PAGE). In females, vitellogenin is indicated by dots. All lanes represented 2.5% of single individuals. EP, early pupa; LL, late larva; LP, late pupal stage; MP, mid-pupal stage; NEA, newly emerged adult; PP, prepupa; S, standard proteins were 45, 66, 97, 116, 200 kDa.

Fig. 3. Native and two-dimensional PAGE showing Plutella xylostella hexamerins. (A) Pore-limited native PAGE shows standards of 669 and 440 kDa and the three hexamerins, indicated by arrowheads. (B) First dimension protein bands in a low density native PAGE rotated 90° counterclockwise. (C) Pattern produced by second dimension SDS–PAGE from first dimension shown in (B). By Western blots, the left two spots were identified as high-methionine hexamerins and the rightmost as arylphorin.

in females and at only 16 µg/mg in males. Just prior to eclosion, levels in females and males dropped to 13 and 5 µg/mg, respectively. During the first 4 h after eclosion, storage protein levels rose dramatically in females. The weight-specific level was similar to the peak concentration in the early pupal period, about 28 µg/mg moth. The total reserve of storage protein dropped from a mean of about 160 to 110 µg. Hexamerin level dropped quickly after the first day of adult life. Egg protein, vitellogenin, was detected in gel samples in a few females as early as the end of the pupal stage (Fig. 2). The level of vitellogenin rose and then maintained a fairly constant level over the first four days of adult life. Eggs were laid as they matured, so vitellogenin did not accumulate.

4. Discussion 4.1. Preliminary characterization of the hexamerins We found three different storage proteins in the diamondback moth, Plutella xylostella. The estimated molecular masses of the three holoproteins and their

Fig. 4. Larger individuals retained a larger percentage of their prepupal weight at emergence than smaller individuals. Females (䊐) were significantly larger than males (쐌).

denatured subunits suggest a hexameric native structure. We identified one of the proteins as an arylphorin, based on its immunoreactivity with an antibody to arylphorin in Manduca sexta, as well its amino acid composition. The other two hexamerins were identified as high-methionine (HM) based on immunoreactivity with two antibodies to HM hexamerins in H. cecropia. In addition, the HM hexamerins were more abundant in females, which is typical in Lepidoptera (Telfer and Kunkel, 1991).

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egg production until conditions are suitable would be advantageous. Although P. xylostella lacks a distinct pre-reproductive period associated with flight (Shirai, 1995) such as that found in several species of noctuid moths (Gatehouse, 1994), resequestration followed by migration would a potential mechanism for an oogenesis-flight syndrome. 4.3. Links between storage proteins and vitellogenesis in other Lepidoptera

Fig. 5. Hexamerins during pupal and adult development in P. xylostella, based on quantitative SDS–PAGE of hexamerin bands (totaled) produced by whole body extracts. Error bars represent standard errors. Lack of bar indicates S.E. ⬍ size of symbol. Stage abbreviations as in Fig. 2. LLP, late late pupae just prior to eclosion; VNEA, very newly emerged adults, immediately after eclosion.

4.2. Amino acid sequestration by Plutella and its implications Levels of storage protein rose dramatically in P. xylostella females immediately after eclosion. This finding, which has not been reported previously in insects, suggests that adult females may synthesize hexamerins to resequester amino acids. The rise is extremely rapid, in contrast to the cyclical rise and fall of hexamerin abundance observed for arylphorin during larval/larval molts in the gypsy moth, Lymantria dispar. In that case, serum arylphorin levels rise until apolysis, fall until ecdysis, and then rise slowly following the molt (Karpells et al., 1990). The role that resequestration plays in this rise, if any, is unknown. Insects that accumulate the vast majority of their eggdestined amino acids during larval development and in which vitellogenin synthesis and egg production takes place after eclosion could be well served by a strategy of resequestration. Once in storage, amino acids could be meted out in response to local environment. The timing of egg development and oviposition in P. xylostella is sensitive to a variety to conditions including sugar feeding, mating, adult densities, and presence of host plants (Hillyer and Thorsteinson, 1969; Pivnick et al., 1990). The capacity to delay vitellogenin synthesis and

The initiation of vitellogenin synthesis in diverse Lepidoptera is known to fall anytime from the last larval instar, as in the gypsy moth, Lymantria dispar (Davis et al., 1990; Lamison et al., 1991), to well after eclosion in the adult stage, as in Heliconius butterflies (Boggs, 1981). Hormonal implications of the timing of vitellogenin synthesis with respect to metamorphosis were recently reviewed by Ramaswamy et al. (1997). Hexamerin synthesis and breakdown is a critical element in the coordination of nutrient intake with nutrient-requiring processes such as metamorphosis and egg production. Here, we review how hexamerins affect the interaction between metamorphosis and egg production, particularly in Lepidoptera. The only insect documented so far to begin synthesis of vitellogenin in the last larval stage is the gypsy moth, Lymantria dispar. L. dispar females begin vitellogenin synthesis on the third day of the last larval instar and continue well into the pupal stage (Lamison et al., 1991). To the extent that vitellogenin synthesis takes place before pupation, it could be supplied by free amino acids derived directly from larval diet and metabolism. Synthesis during the pupal stage may draw on the amino acid pool derived from mobilized arylphorin. Female L. dispar females lack the methionine-rich, female-biased storage proteins found in many Lepidoptera (Lamison et al., 1991). If much of the amino acid demand for vitellogenin synthesis is supplied directly by larval feeding and metabolism, the synthesis of a female-specific store to support egg production may be unnecessary. Larval synthesis of vitellogenin may be part of a syndrome that characterizes insects that show eruptive and cyclical population dynamics. Females of eruptive insects tend to have a reduced flight mechanism and reduced mouthparts, associated with reduced flight and lack of feeding (Miller, 1996; Tammaru and Haukioja, 1996). L. dispar females convert an unusually high percentage of their larval intake to eggs (Montgomery, 1982), perhaps by diverting resources from flight-muscles and mouthparts as well as from the metabolic requirements of synthesizing methionine-rich storage proteins. Silkmoths, which include Bombyx mori (Bombycidae) and Hyalophora cecropia (Saturniidae), and the hawkmoth Manduca sexta (Sphingidae) have been used widely as model systems to explore physiological pro-

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cesses. One feature that has made these insects tractable models is the fact that they mate and lay eggs shortly after eclosion. Adults do not have to be maintained for extended periods under specific conditions in order to propagate laboratory lines. A perhaps unintended result of this feature is that most information known for the regulation of both metamorphosis and egg development in Lepidoptera is based on insects in which the two processes overlap substantially. In the commercial silk moth, Bombyx mori, vitellogenin synthesis begins after pupation. Yolk is deposited into oocytes during pharate adult development and oogenesis is completed before eclosion. During this time, levels of both arylphorin and the methionine-rich storage protein fall to near 0 (Tojo et al., 1980; Ogawa and Tojo, 1981). Hyalophora cecropia and Actias luna, silk moths in the family Saturniidae, show a similar pattern of storage protein depletion, vitellogenin synthesis, and oogenesis. During metamorphosis in Actius luna, both somatic tissue and oocytes draw on amino acids derived from arylphorin and methionine-rich hexamerins (Pan and Telfer, 1996). Vitellogenin is synthesized at two different times. During the pharate pupal stage vitellogenin is synthesized and found in the hemolymph, where it remains during diapause. Vitellogenin is also synthesized during the pupal period in large quantities (Pan, 1971). In Actias luna, vitellogenin synthesized during the earlier pharate pupal period does not incorporate amino acids derived from storage proteins (Pan and Telfer, 1996). This portion of vitellogenin produced may be derived directly from larval diet and metabolism, as in the L. dispar. In Manduca sexta, arylphorin and methionine-rich hexamerin are synthesized during the last larval stage. Expression of arylphorin mRNA stops by the wandering stage (Webb and Riddiford, 1988). Arylphorin accumulates, is present for much of pupal stage, and is depleted before eclosion (Kramer et al., 1980). Methionine-rich hexamerin levels peak just before pupation (Ryan et al., 1985). Vitellogenin first appears in the hemolymph toward the end of the pupal period, four days prior to eclosion. The level is high from the day before the pupal/adult molt through the first five days of adult life (Imboden and Law, 1983). At eclosion, oocytes begin to take up yolk and other egg constituents, then hydrate and produce the chorion (Nijhout and Riddiford, 1974). The likely sources of amino acids used to synthesize vitellogenin are the storage hexamerins, but this has not been verified. Hawkmoths, unlike silk moths, are able to feed; the long-proboscis is evidence of their nectar gathering ability. Recent work with the Nessus Hawkmoth, Amphion floridensis, using stable carbon isotopes to label amino acids and sugars, showed that females assimilated sugars but not free amino acids into eggs. Adult physiology seems to prohibit incorporation of amino acids derived from adult feeding into eggs

(O’Brien, 1998). Vitellogenesis seems to be supported exclusively by amino acids from storage proteins. The ability of adults to feed therefore does not mean that all nutrients consumed by adults are physiologically available. Diverse Lepidoptera both synthesize vitellogenin and produce eggs entirely within the adult stage. Some of these are dependent entirely on stored amino acid reserves while others clearly enhance their fecundity through amino acids and other nutrients obtained from their adult diet or from males during mating (e.g., Boggs, 1997). P. xylostella is a member of this group, but its ability to use dietary amino acids has not been investigated. Some Heliconius butterflies are the best known examples of Lepidoptera that obtain a large proportion of amino acids required for oogenesis from adult feeding as well as male donations (Boggs, 1987). 4.4. Other holometabolous insects using storage proteins to supply female reproduction in the adult stage Females of any insect species that does not acquire protein as an adult but does initiate vitellogenesis must draw on larval reserves. Storage proteins represent one likely source of amino acids for vitellogenesis, but remarkably few cases have been documented. Previously we examined depletion of storage protein reserves followed by appearance of vitellogenin in an autogenous mosquito. Female Aedes atropalpus rarely take blood meals and use reserves accumulated during the larval stage to provision eggs (O’Meara and Krasnick, 1970). As in P. xylostella, a substantial amount of storage protein remains at the end of the pupal stage, but in contrast, the hexamerin store continues to decline steadily after eclosion (Wheeler and Buck, 1996). One important difference between the two species is that A. atropalpus eggs mature in one large synchronous batch over three to four days immediately after the molt to the adult stage. Eclosion in fact triggers the same sequence of steps in egg development that occurs in blood-feeding relatives (Kelly et al., 1981). The tight linkage between eclosion and vitellogenesis means that these mosquitoes do not have a flexible prereproductive period. 4.5. Sequestration of adult-derived amino acids into storage proteins in other insects The amount of storage hexamerins in P. xylostella females rises sharply immediately after eclosion. The rise could be explained by de novo synthesis of storage proteins using amino acids released but not used during metamorphosis. The ability to synthesize and store amino acids as storage proteins during the adult stage, has wide-ranging implications for protein management in insects, particularly those that are long-lived. A sur-

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prising amount of evidence, albeit scattered, supports the conjecture that excess protein obtained by adult females can be stored. For example, adult females in the kissing bug Rhodnius prolixus synthesize storage protein after each engorgement, along with vitellogenin (Chinzei et al., 1994). The viviparous roach, Diploptera punctata, has cycles of pregnancy. Protein granule storage in the fat body is correlated with these cycles; granules disappear during vitellogenesis and reappear during gestation (Stay and Clark, 1971). The granules have a morphology typical of storage proteins (Wang and Haunerland, 1991). In the ant Camponotus festinatus, both queens and workers synthesize storage proteins in response to local conditions, and good circumstantial evidence suggests these support vitellogenesis and feeding of larvae (Martinez and Wheeler, 1994). Rooney and Lewis (1999) recently reported differential allocation of malederived amino acids in two species of lampyrid beetles. Photinus ignitus is short-lived and matures eggs soon after eclosion. Two days after mating, amino acids from the male spermatophore were found predominately (62%) in eggs and less (27%) in somatic tissue. Ellychnia corrusca is longer-lived and produces eggs over several weeks. In this species, by six days after mating, only 22% of the amino acid label was found in oocytes while 46% was located in the fat body. Although hexamerins were not assayed, the results do suggest that E. corrusca stores male-derived amino acids in the fat body for later use. Finally, in a study on the reproductive allocation from juvenile and adult feeding in two species of nymphalid butterflies, tracer studies showed a decline in the use of larval-derived amino acids in eggs over time (Boggs, 1997). Boggs (1997) suggested that one explanation could be that the pool from which larval reserves were drawn was being diluted by storage of adult nutrients. All these cases suggest that amino acids not used immediately for vitellogenesis may be sequestered as storage proteins and reserved for later use. In summary, physiological links between the amino acids stored in storage proteins and those acids used in vitellogenesis may be common in insects. In addition, the dynamics between sequestration and release of amino acids from storage proteins in the adult stage may be an important feature enabling many of their diverse strategies of reproduction.

Acknowledgements This research was supported by the Undergraduate Biology Research Program through BIR 9220332, USDA funds to B.E.T, and Agricultural Station Funds to D.E.W. We thank Susan Meyer for providing a steady supply of cabbage plants and moth eggs.

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